THYROID-HORMONE RECEPTOR BETA-2 PROMOTER ACTIVITY IN PITUITARY-CELLS IS REGULATED BY PIT-1

There are three known thyroid hormone receptor (TR) isoforms that aris e from two distinct alpha and beta gene loci. TR alpha 1 and TR beta 1 mRNAs are found in many tissues, whereas mRNA for the N-terminal TR b eta 2 variant derived from the beta locus is readily detectable only i n the pituitary gland and derived cell sources such as GH3 somatotrope s and TtT-97 thyrotropes. We previously isolated the genomic region go verning expression of the TR beta 2 isoform in thyrotropes and showed that transcription arose from multiple origins within a 400-base pair (bp) region. We now report that the region extending 500 bp upstream o f the putative AUG codon (A is +1) contains six areas of interaction w ith the pituitary-specific transcription factor Pit-1. In addition the re are separate areas that bind other factors present in thyrotrope ce lls. Promoter deletions revealed that removal of regions containing th e Pit-1 sites at -456 to -432, -149 to -127, and -124 to -102 progress ively decreased TR beta 2 promoter activity in thyrotropes. A more pro ximal foot printed area from -65 to -19, which accounted for the remai ning promoter activity, contained sites that interacted with recombina nt Pit-1; however, extracts of TtT-97 thyrotropes, which express Pit-1 , footprinted this proximal region with a pattern of protection that d iffered from that produced by Pit-1. A comparative deletional analysis demonstrated that a shorter region extending only 204 bp from the AUG was sufficient to support TR beta 2 promoter activity in GH3 somatotr opes. The more proximal Pit-1 sites, including the area from -53 to -1 9, whose pattern differed from Pit-1 in thyrotrope extracts, showed pr otection patterns with GH3 extracts that were indistinguishable from r ecombinant Pit-1. Site-directed mutagenesis that abrogated binding of both recombinant Pit-1 and Pit-1-containing nuclear extracts revealed that the two Pit-1 sites between -149 and -102 were important for TR b eta 2 promoter activity with the more proximal being most critical. Fi nally, we showed that TR beta 2 promoter activity in alpha-TSH cells, which do not transcribe the endogenous TR beta 2 locus or produce Pit- 1 protein, could be reconstituted to a level approaching that seen in expressing TtT-97 thyrotropes by cotransfecting a Pit-1 expression vec tor. Activation by Pit-1 was dependent on the same Pit-1 sites shown t o be important for basal TR beta 2 promoter activity in thyrotropes as constructs lacking them by deletion or mutation were not stimulated b y Pit-1.